Construction method of 16srRNA multiplex sequencing library

Abstract

The invention relates to the technical field of molecular biology, and particularly relates to a construction method of a 16SrRNA multiplexed sequencing library. The construction method comprises the following steps: (1) amplification PCR (Polymerase Chain Reaction): forming a primer pair by using a primer F1 and a primer R1, and amplifying a 16SrRNA sequence in a sample by a PCR; (2) library construction PCR: forming a primer pair by using a primer F2 and a primer R2, and carrying out PCR amplification by using a PCR product obtained in the step (1) as a template. According to the construction method of the 16SrRNA multiplexed sequencing library, which is provided by the invention, data obtained by a sequencing reaction of the sequencing library is better in quality and balance, and a sequencing result is more accurate; a number of library sample labels capable of being realized is much greater than that in the prior art; the construction method is more suitable for researching bacterial diversity in a soil sample; accuracy of researching bacterial diversity in the sample is greatly improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a 16SrRNA multiple sequencing library. Background technique [0002] Bacterial diversity is closely related to many fields of human life and production. Understanding and mastering the information of bacterial diversity in different habitats can play an important guiding role in many aspects such as food processing, agricultural production, and medical treatment. With the development of molecular biology techniques, the research methods of bacterial diversity based on 16SrRNA sequences have been continuously improved. This type of method extracts the microbial environmental genome (also called metagenomics, Metagenome, that is, the sum of the genetic material of all tiny organisms in a certain habitat) in an unknown sample, amplifies the 16SrRNA sequence through 16SrRNA primers, and constructs a 16SrRNA sequencing library. After connecting nec...

AI Technical Summary

Problems solved by technology

Through comparative experiments, it was found that the 16SrRNA sequencing library construction primers in the prior art (such as the primers provided by Illunima) can obtain relatively specific amplification results when used in samples such as intestinal tract; but when used in soil samples, the obtained 16SrRNA The specificity of the amplified product is not high, and electrophoresis shows that the amplified bands are seriously diffused, which will seriously affect the accuracy of subsequent sequencing and analysis results

[0006] On the other hand, although existing technologies have increased the number of different library samples that can be mixed and sequenced in multiplex sequencing, as research needs continue to increase, the number of samples that need to be mixed and sequenced is also increasing

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